1. Field of the Invention
The invention relates generally to evanescent wave-type biosensors, or biomolecular assays. More specifically, the invention relates to biosensors including substrates with metallic films on one or more surfaces thereof and, in particular, to biosensors with metallic films that include nanocavities with shapes that are configured to optimize the amplification of signals indicative of the presence or amount of one or more analytes present in a sample.
2. Background of Related Art
Plasmonics is the study of phenomena related to the interaction of electromagnetic radiation with an electron gas (or plasma) at a metal surface (B. Schechter “Bright new world,” New Scientist 31-33 (2003)). Aside from the now-common surface plasmon resonance (SPR)-based sensors (B. Liedberg, C. Nylander, and I. Lundstrom, “Surface plasmon resonance for gas detection and biosensing,” Sen. Actuators, vol. 4, pp. 299-304, 1983; N. Bianchi, C. Rustigliano, M. Tomassetti, G. Feriotto, F. Zorzato, and R. Gambari, “Biosensor technology and surface plasmon resonance for real-time detection of HIV-1 genomic sequences amplified by polymerase chain reaction,” Clin. Diagnostic Virology, vol. 8, pp. 199-208, 1997), plasmonics has been applied to molecular detection applications by attaching metallic nanoparticles to molecules for use as light scattering labels (J. Yguerabide and E. E. Yguerabide, “Light-scattering submicroscopic particles as highly fluorescent analogs and their use as tracer labels in clinical and biological applications,” Anal. Biochem., vol. 262, no. 2, pp. 157-176, September 1998; T. A. Taton, C. A. Mirkin, and R. L. Letsinger, “Scanometric DNA array detection with nanoparticle probes,” Science, vol. 289, pp. 1757-1760, 2000; L. R. Hirsch, J. B. Jackson, A. Lee, N. J. Halas, and J. L. West, “A whole blood immunoassay using gold nanoshells,” Anal. Chem., vol. 75, p. 2377, 2003) in biosensing. Nanostructured metallic surfaces have also been studied extensively for surface-enhanced fluorescence (A. Wokaun, H.-P. Lutz, A. P. King, U. P. Wild, and R. R. Ernst, “Energy transfer in surface enhanced luminescence,” J. Chem. Phys., vol. 79, no. 1, pp. 509-514, 1983; J. Malicka, I. Gryczynski, Z. Gryczynski, and J. R. Lakowicz, “Effects of fluorophore-to-silver distance on the emission of cyanine-dye-labeled oligonucleotides,” Anal. Biochem., vol. 315, pp. 57-66, 2003) and Raman scattering (SERS) (K. Kneipp, H. Kneipp, R. Manoharan, E. B. Hanlon, I. Itzkan, R. R. Dasari, and M. S. Feld, “Extremely large enhancement factors in surface-enhanced Raman scattering for molecules on colloidal gold clusters,” Appl. Spectros., vol. 52, pp. 1493-1497, 1998). One of the major drawbacks of these surface-enhanced techniques is that the nanostructure is disordered (but sometimes with fractal order) such that the fluorescence or Raman enhancement factors are spatially-varying, as evidenced by “hot-spots” on the surface (V. M. Shalaev, R. Botet, J. Mercer, and E. B. Stechel, “Optical properties of self-affine thin films,” Phys. Rev. B, vol. 54, pp. 8235-8242, 1996). The hot-spot effect may render these techniques unsuitable for quantitative assays, especially in an array format, as the average enhancement over a defined sensing zone may not be very high, and the enhancement from zone to zone may vary. As a result, there have been efforts in which molecules are attached to lithographically defined arrays of metallic nanoparticles (H. Ditlbacher, N. Felidj, J. R. Krenn, B. Lambprecht, A. Leitner, and F. R. Aussenegg, “Electromagnetic interaction of fluorophores with designed 2D silver nanoparticle arrays,” Appl. Phys. B, vol. 73, p. 373, 2001; N. Felidj, J. Aubard, G. Levi, J. R. Krenn, A. Hohenau, G. Schider, A. Leitner, and F. R. Aussenegg, “Optimized surface-enhanced Raman scattering on gold nanoparticle arrays,” Appl. Phys. Lett., vol. 82, no. 18, pp. 3095-3097, 2003). With these architectures, uniformity in nanoparticle size, shape, and spacing result in well-defined enhancement in terms of magnitude and spatial location. However, these techniques do not provide complete isolation from background produced by unbound species, as uniform illumination can excite fluorescence from molecules located between nanoparticles, which produce background signals at the detector.
An important recent advance is the demonstration of extraordinary light transmission through a periodic array of subwavelength metallic apertures (T. W. Ebbeson, H. J. Lezec, H. F. Ghaemi, T. Thio, and P. A. Wolff, “Extraordinary optical transmission through sub-wavelength hole arrays,” Nature, vol. 391, pp. 667-669, 1998) or nanocavities, where, in the absence of the nanocavities, no light passes through the metal film. Even though this has been quite an active area of research, some disagreement about the origin of the transmission enhancement still exists (H. J. Lezec and T. Thio, “Diffracted evanescent wave model for enhanced and suppressed optical transmission through subwavelength hole arrays,” Optics Express, vol. 12, no. 16, pp. 3629-3651, 2004). However, it is generally believed (I. Avrutsky, Y. Zhao, and V. Kochergin, “Surface-plasmon-assisted resonant tunneling of light through a periodically corrugated thin metal film,” Opt. Lett., vol. 25, pp. 595-597, 2000; A. K. Sarychev, V. A. Podolsky, A. M. Dykhne, and V. M. Shalaev, “Resonance transmittance through a metal film with subwavelength holes,” IEEE J. Quantum Electron., vol. 38, pp. 956-963, 2002; L. Martin-Moreno and F. J. Garcia-Vidal, “Theory of extraordinary optical transmission through subwavelength hole arrays,” Opt. Express, vol. 12, pp. 3619-3628, 2004.5) that the periodic array of nanocavities acts as a two-dimensional diffraction grating, which, at specific incidence angles, allows light to couple from free space into surface plasmon polariton (SPP) Bloch modes on either metal interface. These SPP modes can constructively interfere within the nanocavities, resulting in intensity enhancement (H. J. Lezec and T. Thio, “Diffracted evanescent wave model for enhanced and suppressed optical transmission through subwavelength hole arrays,” Optics Express, vol. 12, no. 16, pp. 3629-3651, 2004) and, therefore, greater transmission. The inventors have demonstrated experimentally, using fluorophores as local intensity probes, that light is indeed localized within the nanocavities (Y. Liu and S. Blair, “Fluorescence enhancement from an array of subwavelength metal apertures,” Opt. Lett., vol. 28, pp. 507-509, 2003) and that enhanced fluorescence transduction can be performed (Y. Liu, J. Bishop, L. Williams, S. Blair, and J. N. Herron, “Biosensing based upon molecular confinement in metallic nanocavity arrays,” Nanotechnology, vol. 15, pp. 1368-1374, 2004; Y. Liu and S. Blair, “Fluorescence transmission through 1-D and 2-D periodic metal films,” Opt. Express, vol. 12, no. 16, pp. 3686-3693, 2004).
More recently, enhancement in single molecule fluorescence h as been reported for round (H. Rigneault, J. Capoulade, J. Ditinger, J. Wenger, N. Bonod, E. Popov, T. W. Ebbesen, and P.-F. Lenne, “Enhancement of single-molecule fluorescence detection in subwavelength apertures,” Physical Review Letters 95, 117401 (2005)) and rectangular (J. Wenger, P.-F. Lenne, E. Popov, H. Rigneault, J. Ditinger, and T. W. Ebbesen, “Single-molecule fluorescence in rectangular nano-apertures,” Optics Express 13, 7035-7044 (2005)) nanoapertures, and a computational model for radiative enhancement has been developed (Y. Liu, F. Mandavi, and S. Blair, “Enhanced fluorescence transduction properties of metallic nanocavity arrays,” IEEE Journal of Selected Topics in Quantum Electronics 11, 778-784 (2005)).
Multi-analyte, or array, biosensing is an increasingly important area of research and development for many clinical, environmental, and industrial applications. In the clinical application of genetic screening, for example, high sensitivity hybridization arrays are needed for rapid identification of genetic disorders in the presence of multiple genotypes or mutations (B. J. Maron, J. H. Moller, C. E. Seidman, G. M. Vincent, H. C. Dietz, A. J. Moss, H. M. Sondheimer, R. E. Pyeritz, G. McGee, and A. E. Epstein, “Impact of laboratory molecular diagnosis on contemporary diagnostic criteria for genetically transmitted cardiovascular diseases: hypertrophic cardiomyopathy, long-QT syndrome, and Marfan syndrome,” Circulation 98, 1460-1471 (1998); J. G. Hacia “Resequencing and mutational analysis using oligonucleotide microarrays,” Nature Genetics 21, 42-47 (1999)).
However, many challenges, such as improving sensitivity, accuracy, precision and specificity of the assays, reducing assay time, etc., remain in the field.